Validation of an ELISA test kit for the detection of ochratoxin A in several food commodities by comparison with HPLC

Abstract

An ELISA Microtiter Plate, Ochratoxin Test called AgraQuant was validated to measure ochratoxin A in a range from 2 to 40 ppb in corn, milo, barley, wheat, soybeans and green coffee. The test is performed as a solid phase direct competitive ELISA using a horseradish peroxidase conjugate as the competing, measurable entity. For the test method, ochratoxin A is extracted from ground samples with 70% methanol and sample extracts plus conjugate are mixed and then added to the antibody-coated microwells. After 10 min incubation at room temperature, the plate is washed and enzyme substrate is added and allowed to incubate for an additional 5 min. Stop solution is then added and the intensity of the resulting yellow color is measured optically with a microplate reader at 450 nm. Results obtained from internal validation studies assessing accelerated stability indicate a 1 year shelf life; accuracy and precision are comparable to HPLC from 0 to 80 ppb and limit of detection in corn is 1.9 ppb and other food commodities is up to 3.8 ppb. Comparison of the method to HPLC, ability to detect individual ochratoxins, and ruggedness of the test kits determined this test to be rugged from 18 to 30 degrees C, sensitive, accurate, precise and effective comparable to HPLC for measuring ochratoxin A ranging from 2 to 40 ppb in several commodities.